Tumor necrosis factor-.alpha. (TNF-.alpha., also known as cachectin) is a mammalian protein capable of inducing a variety of effects on numerous cell types. TNF-.alpha. was initially characterized by its ability to cause lysis of tumor cells and is produced by activated cells such as mononuclear phagocytes, T-cells, B-cells, mast cells and NK cells. In mononuclear phagocytes, TNF-.alpha. is initially synthesized as a membrane-bound protein of approximately 26 kD. A 17 kD fragment of the 26 kD membrane-bound TNF-.alpha. is "secreted" and combines with two other secreted TNF-.alpha. molecules to form a circulating 51 kD homotrimer. TNF-.alpha. is a principal mediator of the host response to gram-negative bacteria. Lipopolysaccharide (LPS, also called endotoxin), derived from the cell wall of gram-negative bacteria, is a potent stimulator of TNF-.alpha. synthesis. Because the deleterious effects which can result from an over-production or an unregulated-production of TNF are extremely serious, considerable efforts have been made to control or regulate the serum level of TNF. An important part in the effort to effectively control serum TNF levels is the understanding of the mechanism of TNF biosynthesis.
The mechanism by which TNF-.alpha. is secreted has only been recently elucidated. Kriegler et al. Cell, 53, 45-53, (1988) conjectured that TNF-.alpha. "secretion" is due to the cleaving of the 26 kD membrane-bound molecule by a proteolytic enzyme or protease. Scuderi et. al., J. Immunology, 143, 168-173 (1989), suggested that the release of TNF-.alpha. from human leukocyte cells is dependent on one or more serine proteases, e.g., a leukocyte elastase or trypsin. A serine protease inhibitor, p-toluenesulfonyl-L-arginine methyl ester, was found to suppress human leukocyte TNF release in a concentration-dependent manner. Scuderi et. al. suggested that the arginine methyl ester competes for the arginine-binding site in the enzyme's reactive center and thereby blocks hydrolysis. The lysine and phenylalanine analogs of the inhibitor reportedly failed to mimic the arginine methyl ester.
We have discovered that the protease which causes the cleavage of the TNF-.alpha. molecule into the 17 kD protein is, in fact, a metalloprotease which is believed to reside in the plasma membrane of cells producing TNF-.alpha.. The physicochemical characteristics of the enzyme have not been published.
Most, but not all, proteases recognize a specific amino acid sequence. Some proteases primarily recognize residues located N-terminal of the cleaved bond, some recognize residues located C-terminal of the cleaved bond, and some proteases recognize residues on both sides of the cleaved bond. Metalloprotease enzymes utilize a bound metal ion, generally Zn.sup.2+, to catalyze the hydrolysis of the peptide bond. Metalloproteases are implicated in joint destruction (the matrix metalloproteases), blood pressure regulation (angiotensin converting enzyme), and regulation of peptide-hormone levels (neutral endopeptidase-24.11).
Numerous inhibitors have been developed against the previously described metalloproteases. A general family of inhibitors against matrix-metalloproteases, and in particular collagenase, is reported in WO 92/09563. This document shows compounds having the general structure of a reverse hydroxamate--or a hydroxyurea--linked via an amide to an amino acid derivative, such as tryptophan or 2-naphthyl alanine. Inhibitors of collagenase are also reported in WO 88/06890; these compounds contain sulfhydryl moieties as well as phenylalanine and tryptophan analogs. Collagenase inhibitors are reported in WO 92/09556 and U.S. Pat. No. 5,114,953 and possess hydroxamate moities and fused or conjugated bicycloaryl substituents. The myriad potential gelatinase inhibitors covered by the generic formula in EPA 489,577 are amino acid derivatives optionally possessing a hydroxamate group. Hydroxamate derivatives useful as angiotensin converting enzyme (ACE) inhibitors are reported in EPO 498,665.
Inhibition of the TNF-.alpha. converting enzyme (hereinafter referred to as "TACE"), a novel metalloprotease, inhibits release of TNF-.alpha. into the serum and other extracellular spaces. TACE inhibitors would therefore have clinical utility in treating conditions characterized by over-production or unregulated production of TNF-.alpha.. A particularly useful TACE inhibitor for certain pathological conditions would selectively inhibit TACE while not affecting TNF-.beta. (also known as lymphotoxin) serum levels. The over-production or unregulated production of TNF-.alpha. has been implicated in certain conditions and diseases, for example:
I. Systemic Inflammatory Response Syndrome, which includes: PA0 II. Reperfusion Injury, which includes: PA0 III. Cardiovascular Disease, which includes: PA0 IV. Infectious Disease, which includes: PA0 V. Obstetrics/Gynecology, including: PA0 VI. Inflammatory Disease/Autoimmunity, which includes: PA0 VII. Allergic/Atopic Diseases, which includes: PA0 VIII. Malignancy, which includes: PA0 IX. Transplants, including: PA0 X. Cachexia PA0 XI. Congenital, which includes: PA0 XII. Dermatologic, which includes: PA0 XIII. Neurologic, which includes: PA0 XIV. Renal, which includes: PA0 XV. Toxicity, which includes: PA0 XVI. Metabolic/Idiopathic, which includes:
Sepsis syndrome PA1 Trauma/hemorrhage PA1 Burns PA1 Ionizing radiation exposure PA1 Acute pancreatitis PA1 Adult respiratory distress syndrome. PA1 Post pump syndrome PA1 Ischemia-reperfusion injury PA1 Cardiac stun syndrome PA1 Myocardial infarction PA1 Congestive heart failure PA1 HIV infection/HIV neuropathy PA1 Meningitis PA1 Hepatitis PA1 Septic arthritis PA1 Peritonitis PA1 Pneumonia PA1 Epiglottitis PA1 E. coli 0157:H7 PA1 Hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura PA1 Malaria PA1 Dengue hemorrhagic fever PA1 Leishmaniasis PA1 Leprosy PA1 Toxic shock syndrome PA1 Streptococcal myositis PA1 Gas gangrene PA1 Mycobacterium tuberculosis PA1 Mycobacterium avium intracellulare PA1 Pneumocystis carinii pneumonia PA1 Pelvic inflammatory disease PA1 Orchitis/epidydimitis PA1 Legionella PA1 Lyme disease PA1 Influenza A PA1 Epstein-Barr Virus PA1 Viral-associated hemaphagocytic syndrome PA1 Viral encephalitis/aseptic meningitis PA1 Premature labor PA1 Miscarriage PA1 Infertility PA1 Rheumatoid arthritis/seronegative arthropathies PA1 Osteoarthritis PA1 Inflammatory bowel disease PA1 Systemic lupus erythematosis PA1 Iridocyclitis/uveitis/optic neuritis PA1 Idiopathic pulmonary fibrosis PA1 Systemic vasculitis/Wegener's granulomatosis PA1 Sarcoidosis PA1 Orchitis/vasectomy reversal procedures PA1 Asthma PA1 Allergic rhinitis PA1 Eczema PA1 Allergic contact dermatitis PA1 Allergic conjunctivitis PA1 Hypersensitivity pneumonitis PA1 ALL PA1 AML PA1 CML PA1 CLL PA1 Hodgkin's disease, non-Hodgkin's lymphoma PA1 Kaposi's sarcoma PA1 Colorectal carcinoma PA1 Nasopharyngeal carcinoma PA1 Malignant histiocytosis PA1 Paraneoplastic syndrome/hypercalcemia of malignancy PA1 Organ transplant rejection PA1 Graft-versus-host disease PA1 Cystic fibrosis PA1 Familial hematophagocytic lymphohistiocytosis PA1 Sickle cell anemia PA1 Psoriasis PA1 Alopecia PA1 Multiple sclerosis PA1 Migraine headache PA1 Nephrotic syndrome PA1 Hemodialysis PA1 Uremia PA1 OKT3 therapy PA1 Anti-CD3 therapy PA1 Cytokine therapy PA1 Chemotherapy PA1 Radiation therapy PA1 Chronic salicylate intoxication PA1 Wilson's disease PA1 Hemachromatosis PA1 Alpha-1-antitrypsin deficiency PA1 Diabetes PA1 Hashimoto's thyroiditis PA1 Osteoporosis PA1 Hypothalamic-pituitary-adrenal axis evaluation PA1 Primary biliary cirrhosis PA1 m is 0, 1 or 2; PA1 R.sup.1, R.sup.2 and R.sup.3 each independent of the other is hydrogen, alkylene(cycloalkyl), OR.sup.4, SR.sup.4, N(R.sup.4)(R.sup.5), halogen, substituted or unsubstituted C.sub.1 to C.sub.8 alkyl, C.sub.1 to C.sub.8 alkylenearyl, aryl, a protected or unprotected side chain of a naturally occurring .alpha.-amino acid; or the group --R.sup.6 R.sup.7, wherein R.sup.6 is substituted or unsubstituted C.sub.1 to C.sub.8 alkyl and R.sup.7 is OR.sup.4, SR.sup.4, N(R.sup.4)(R.sup.5) or halogen, wherein R.sup.4 and R.sup.5 are, each independent of the other, hydrogen or substituted or unsubstituted C.sub.1 to C.sub.8 alkyl; PA1 n is 0, 1 or 2; PA1 provided that when n is 1, A is a protected or an unprotected .alpha.-amino acid radical; PA1 when n is 2, A is the same or different protected or unprotected .alpha.-amino acid radical; and PA1 B is unsubstituted or substituted C.sub.2 to C.sub.8 alkylene; PA1 and the pharmaceutically acceptable salts thereof. PA1 m is 0, 1 or 2; PA1 R.sup.1, R.sup.2 and R.sup.3 each independent of the other is hydrogen, alkylene(cycloalkyl), OR.sup.4, SR.sup.4, N(R.sup.4)(R.sup.5), halogen, substituted or unsubstituted C.sub.1 to C.sub.8 alkyl, C.sub.1 to C.sub.8 alkylenearyl, aryl, a protected or unprotected side chain of a naturally occurring .alpha.-amino acid; or the group --R.sup.6 R.sup.7, wherein R.sup.6 is C.sub.1 to C.sub.8 alkyl and R.sup.7 is OR.sup.4, SR.sup.4, N(R.sup.4)(R.sup.5) or halogen, wherein R.sup.4 and R.sup.5 are each, independent of the other, hydrogen or substituted or unsubstituted C.sub.1 to C.sub.8 alkyl; PA1 n is 0, 1 or 2; PA1 Y is hydrogen, unsubstituted or substituted C.sub.1 to C.sub.8 alkyl, alkylene(cycloalkyl), the group --R.sup.8 --COOR.sup.9 or the group --R.sup.10 N(R.sup.11)(R.sup.12); wherein R.sup.8 is C.sub.1 to C.sub.8 alkylene; PA1 R.sup.9 is hydrogen or C.sub.1 to C.sub.8 alkyl; R.sup.10 is unsubstituted or substituted C.sub.1 to C.sub.8 alkylene; and R.sup.11 and R.sup.12 are each, independent of the other, hydrogen or C.sub.1 to C.sub.8 alkyl; PA1 provided that when n is 1, A is a protected or an unprotected .alpha.-amino acid radical; and PA1 when n is 2, A is the same or different protected or unprotected .alpha.-amino acid radical; PA1 and the pharmaceutically acceptable salts thereof; PA1 wherein the compound is capable of reducing serum TNF-.alpha. levels by at least 80% when administered at 25 mg/kg in a murine model of LPS-induced sepsis syndrome; PA1 and a pharmaceutically acceptable carrier. PA1 m is 0, 1 or 2; PA1 R.sup.1, R.sup.2 and R.sup.3 each independent of the other is hydrogen, alkylene(cycloalkyl), OR.sup.4, SR.sup.4, N(R.sup.4)(R.sup.5), halogen, substituted or unsubstituted C.sub.1 to C.sub.8 alkyl, C.sub.1 to C.sub.8 alkylenearyl, aryl, a protected or unprotected side chain of a naturally occurring .alpha.-amino acid; or the group --R.sup.6 R.sup.7, wherein R.sup.6 is substituted or unsubstituted C.sub.1 to C.sub.8 alkyl and R.sup.7 is OR.sup.4, SR.sup.4, N(R.sup.4)(R.sup.5) or halogen, wherein R.sup.4 and R.sup.5 are each, independent of the other, hydrogen or substituted or unsubstituted C.sub.1 to C.sub.8 alkyl; PA1 n is 0, 1 or 2; PA1 provided that when n is 1, A is a protected or an unprotected .alpha.-amino acid radical; PA1 when n is 2, A is the same or different protected or unprotected .alpha.-amino acid radical; and PA1 B is unsubstituted or substituted C.sub.2 to C.sub.8 alkylene; PA1 and the pharmaceutically acceptable salts thereof. PA1 m is 0, 1 or 2; PA1 R.sup.1, R.sup.2 and R.sup.3 each independent of the other is hydrogen, alkylene(cycloalkyl), OR.sup.4, SR.sup.4, N(R.sup.4)(R.sup.5), halogen, substituted or unsubstituted C.sub.1 to C.sub.8 alkyl, C.sub.1 to C.sub.8 alkylenearyl, aryl, a protected or unprotected side chain of a naturally occurring .alpha.-amino acid; or the group --R.sup.6 R.sup.7, wherein R.sup.6 is C.sub.1 to C.sub.8 alkyl and R.sup.7 is OR.sup.4, SR.sup.4, N(R.sup.4)(R.sup.5) or halogen, wherein R.sup.4 and R.sup.5 are each, independent of the other, hydrogen or substituted or unsubstituted C.sub.1 to C.sub.8 alkyl; PA1 n is 0, 1 or 2; PA1 Y is hydrogen, unsubstituted or substituted C.sub.1 to C.sub.8 alkyl, alkylene(cycloalkyl), the group --R.sup.8 --COOR.sup.9 or the group --R.sup.10 N(R.sup.11)(R.sup.12); wherein R.sup.8 is C.sub.1 to C.sub.8 alkylene; PA1 R.sup.9 is hydrogen or C.sub.1 to C.sub.8 alkyl; R.sup.10 is unsubstituted or substituted C.sub.1 to C.sub.8 alkylene; and R.sup.11 and R.sup.12 are each, independent of the other, hydrogen or C.sub.1 to C.sub.8 alkyl; PA1 provided that when n is 1, A is a protected or an unprotected .alpha.-amino acid radical; and PA1 when n is 2, A is the same or different protected or unprotected .alpha.-amino acid radical; PA1 and the pharmaceutically acceptable salts thereof; PA1 wherein the compound is capable of reducing serum TNF levels by at least 80% when administered at 25 mg/kg in a murine model of LPS-induced sepsis syndrome; PA1 and a pharmaceutically acceptable carrier.
gram positive sepsis PA2 gram negative sepsis PA2 culture negative sepsis PA2 fungal sepsis PA2 neutropenic fever PA2 urosepsis PA2 meningococcemia
Inhibitors of TACE would prevent the cleavage of cell-bound TNF-.alpha. thereby reducing the level of TNF-.alpha. in serum and tissues. Such inhibitors would be of significant clinical utility and could be potential therapeutics for treating the above TNF-.alpha.-related disorders.